This invention is directed to a high throughput screening (HTS) method that uses aptamers to facilitate the identification of non-aptamer ligands, typically small molecules. Aptamers can be identified that bind to a wide variety of targets and can be used to identify small molecules that can compete with the aptamers for binding to the target.
Drug discovery in most pharmaceutical companies is heavily focused on the identification of orally active compounds. High throughput screening (HTS) of appropriate libraries of compounds (generally small molecules) against validated targets constitutes one of the major activities in discovery research groups whose overall goal is to create and/or maintain a pipeline of new drug candidates. The development of HTS assays that allow efficient detection of active compounds from such libraries is thus a critically important component of drug discovery. With increasing numbers of both validated targets and compound libraries, HTS laboratories are under considerable pressure to find ways to increase throughput and lower cost. Assay development and validation is often one of the bottlenecks in this process (Fox et al., (November 1998) Drug Discovery and Development (Supplement to RandD Magazine) p. 32-37, incorporated by reference in its entirety).
Aptamers (also termed nucleic acid ligands) are structurally unique nucleic acids capable of binding other molecules (i.e., targets) with high affinity and specificity. Aptamers are derived from large random libraries by the SELEX process in which iterative rounds of affinity selection and amplification are used to identify sequences with specific binding properties. The SELEX process is described in more detail below. To date, aptamers that bind to small organic molecules, carbohydrates, amino acids, peptides and proteins have been identified, illustrating the multitude of binding specificities present in large collections of single stranded nucleic acid sequences (Gold, L., et al. (1995) Annu. Rev. Biochem. 64, 763-797). Aptamers bind to protein targets including growth factors, enzymes, receptors and structural proteins in a highly specific manner and with dissociation constants typically in the nanomolar (and sometimes picomolar) range. Given these unique binding properties, nuclease stabilized and appropriately formulated aptamers have substantial potential as therapeutics. For therapeutic uses, aptamers, like antibodies, are delivered parenterally because of their limited oral availability. The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,475,096, and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as aptamers or nucleic acid ligands, each ligand having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
The SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems. SELEX or SELEX-like processes can be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target. In theory, within a candidate mixture of approximately 1013 to 1018 nucleic acids, at least one nucleic acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describes the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, now abandoned, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d now U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d now abandoned (see U.S. Pat. No. 5,660,985), that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, now abandoned, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d now U.S. Pat. No. 5,637,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexes.xe2x80x9d now U.S. Pat. No. 6,011,020. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The present invention describes the use of aptamers to facilitate the identification of non-aptamer ligands. More specifically, the present invention includes the use of aptamers in competition binding assays to rapidly identify compounds that are capable of displacing the aptamers from their targets. The affinities of competitor compounds can be calculated from the known affinity of the aptamer for its target and the competition profiles. The method is highly versatile and compatible with a variety of HTS platforms since aptamers, as chemically synthesized molecules, can be labeled in a variety of ways without compromising their binding affinity.
The method of the invention is illustrated with two protein targets: platelet derived growth factor (PDGF) and wheat germ agglutinin (WGA). For each protein, a small biased set of molecules is screened for their ability to displace the cognate aptamer: naphthalene sulfonic acid derivatives for PDGF and oligosaccharides for WGA. For both PDGF and WGA, best ligands can be identified readily. Furthermore, binding affinities of the competitors correlate with their activities in in vitro assays (infra, and in U.S. Pat. No. 5,780,222 issued Jul. 14, 1998).